Project Summary Loss of meniscus function can lead to articular cartilage degeneration and osteoarthritis. This poses a clinical challenge in patients who are too young for total knee arthroplasty. To address the clinical need for a more effective treatment for meniscus deficiency, this training project is designed to facilitate a long-term career goal of creating a mesenchymal stem cell (MSC) seeded meniscus scaffold construct that emulates native meniscus biology and function. This project will evaluate the effect of hypoxia, HIF-1a signaling, and particulate oxygen generating systems on the differentiation of human mesenchymal stem cells seeded on a porous allograft derived human meniscus. The biologic characteristics of the cultivated scaffolds will be evaluated and compared with the properties of normal meniscus tissue using quantitative PCR, confocal microscopy, and proteoglycan synthesis analysis. HIF-1a signaling will be upregulated and downregulated using chemical and molecular techniques to elucidate the role of HIF-1a in mediating fibrochondrocyte homeostasis and differentiation in a hypoxia culture environment. This proposal is part of a long term project to create a biologically based meniscus replacement construct with improved long term results compared with allograft meniscus transplant. The studies described in this proposal will: 1) Improve our understanding of the biology of normal adult human meniscus tissue and the role of the transcription HIF-1a in mediating the effect of hypoxia on meniscus cell homeostasis and phenotype. 2) Result in a better understanding of the program of stem cell differentiation toward the fibrochondrocyte phenotype and the role of regulated hypoxia as a bioreactor variable for fibrocartilage tissue engineering. 3) Investigate the impact of hypoxia and particulate oxygen generating systems on the proteoglycan production of tissue engineered meniscus replacement constructs. Taken together, the information gathered from these studies will provide physicians with a better understanding of normal meniscus biology, homeostasis, and differentiation mechanisms. Success in this realm of tissue engineering could lead to alternative and expanded treatments for knee pain related to meniscus pathology. As a physician scientist, this training grant will help me further advance my molecular biology skills and research training by approaching tissue engineering strategies from a mechanism based molecular approach and lay the groundwork for validating the effectiveness of future tissue engineering technologies in the clinical arena.